An efficient protocol for in vitro regeneration of peanut (Arachis hypogaea L ) cultivar L14

The present work aims to establish an efficient protocol for in vitro regeneration of peanut (Arachis hypogaea) cultivar L14. The study showed that de-embryonated cotyledon was a suitable explant for shoot multiplication on MS medium containing 4 mg/L BAP. The highest number of shoots per explant obtained after 4 weeks of culture was up to 6.8 shoots. Shoots in vitro were able to produce a large number of approximately 11 roots on MS medium supplemented with 0.5 mg/L NAA. These results will be very useful in establishing an in vitro regeneration protocol for peanut cultivar L14 during gene transfer in the next studies to improve their disease resistance.

Micropropagation of Chapada Diamantina ornamental bromeliad / Micropropagação de bromélia ornamental da Chapada Diamantina

ABSTRACT: Sincoraea mucugensis (Wand. & A.A. Conc.) LOUZADA & WAND, an endangered bromeliad, is confined to the central region of the Chapada Diamantina, in the municipality of Mucugê, Brazil. From various researches, it is evident that for the propagation of this species, the in vitro technique is a feasible option. However, due to the low multiplication rates reported in various papers, this study aimed to establish a micropropagation protocol of direct organogenesis for S. mucugensis. First, the inoculation of the stem explants was done in MS ½ culture medium which contained different levels of BAP (0.00; 6.66; 8.88; 11.10; 13.20 µM) and NAA (0.00; 2.60; 5.20 µM). These shoots were then subjected to a couple of distinct rooting periods (of 30- and 60-day duration) using activated charcoal; finally, these microplants were transferred to a greenhouse for acclimatization, and covered with transparent plastic cups, as a water loss prevention test method. All the data were submitted to the analysis of variance (ANOVA), and the means were subjected to regression analysis or compared using the Tukey test. The findings revealed that the S. mucugensis stem explants raised in the NAA-rich medium (6.42 to 7.43 shoots/explants) showed high multiplication rates; the shoot rooting was done for 30 days using activated charcoal with the medium. Acclimatization, which was performed by directly exposing the microplants to the ex vitro environment, showed 95% survival rate.

RESUMO: Sincoraea mucugensis (Wand. & A.A. Conc.) LOUZADA & WAND é uma bromélia vulnerável de ocorrência restrita ao município de Mucugê, Chapada Diamantina. Estudos indicam que a cultura de tecidos é uma alternativa viável para a propagação in vitro desta espécie. Contudo, em função das baixas taxas de multiplicação obtidas em estudos anteriores, objetivou-se estabelecer um protocolo de micropropagação via organogênese direta para S. mucugensis. Explantes caulinares foram inoculados em meio de cultura MS ½ contendo diferentes concentrações de BAP (0,00; 6,66; 8,88; 11,10; 13,20 µM) e ANA (0,00; 2,60; 5,20 µM). Os brotos obtidos foram submetidos a diferentes períodos de enraizamento (30 e 60 dias) com carvão ativado; posteriormente as microplantas foram aclimatizadas em casa de vegetação, testando-se o efeito da cobertura com copos plásticos transparentes como estratégias contra perda de água. Os dados foram submetidos à análise de variância (ANOVA) e as médias analisadas por regressão ou comparadas pelo Teste de Tukey. Os resultados demonstram que altas taxas de multiplicação de S. mucugensis são geradas a partir de explantes caulinares cultivados em meio contendo ANA (6,42 a 7,43 brotos/explantes); o enraizamento dos brotos é realizado por 30 dias em meio com carvão ativado e a aclimatização é feita com exposição direta das microplantas ao ambiente ex vitro com 95% de sobrevivência.

A novel cost-effective medium for in vitro cultivation of Gentiana kurroo Royle using sugarcane bagasse

Objective: To develop a simple, cost-effective, and efficient medium by using sugarcane bagasse (SB) as abase material to replace the conventional Murashige and Skoog (MS) medium.Materials and Methods: Water extracts of SB along with some macronutrients and plant growth regulators weregelled with 0.7% agar-agar powder. Nodal segments of Gentiana kurroo were used as explants and inoculatedin the medium and placed in a growth chamber under standard conditions of light and temperature. Out of thetested combinations of plant growth regulators, 0.5 mg/l each of kinetin (KN) and 6-Benzylaminopurine (BAP)showed the excellent shoot multiplication and proliferation rate on the bagasse medium with the same potentialas on the MS medium with an average of 5–6 shoots/explant. In vitro rooting was obtained on half strength MSmedium supplemented with IBA (0.5 mg/l) with an average length of 7–8 cm and 20–25 roots/explant. Theplants were hardened in a mixture of clay loam and farmyard manure in 1:1(w/w) with 70%–80% survival ratewithout any phenotypic aberrations.Conclusion: The results from the present investigation indicate that SB can be used as a cost-effective substituteof MS medium for in vitro propagation of G. kurroo.

In vitro propagation of chia (Salvia hispanica L.) and assessment of genetic fidelity using random amplified polymorphic DNA and intersimple sequence repeat molecular markers

A well-organized micropropagation protocol has been designed for Salvia hispanica L., which bears high nutritional and medicinal value. Seeds of S. hispanica L. were germinated aseptically on half strength MS medium. Nodal explants obtained from in vitro germinated seedling were cultured on MS medium fortified with 6-benzyladenine (BAP) (1–5 mg/l) or Kinetin (Kin) (1–5 mg/l) individually or with α-naphthalene acetic acid (0.1–1 mg/l) and indole3-acetic acid (IAA) (0.1–1 mg/l) for clonal propagation. It was observed that maximum amount of shoots per explant (9.02 ± 2.65) was achieved on culture medium fortified with 3 mg/l BAP which was also optimum for subculturing of the regenerated shoots. Rooting was achieved on medium supplemented with 1 mg/l IBA. The rooted plantlets were acclimatized and transferred to field conditions, with 75% survival rate. Genetic fidelity studies were carried out on regenerated plantlets by 30 random amplified polymorphic DNA and 10 intersimple sequence repeat (ISSR) as molecular markers

Influence of silver nitrate and glutamine on in vitro organogenesis of Lota Bhot (Capsicum chinense Jacq.), an indigenous pungent pepper variety of Assam

Northeast India including Assam is one of the richest centers of pepper diversity. Chilli peppers are valued commercially owing to their pungency ingredient, capsaicin. Capsicum chinense variety Lota Bhot is an extremely pungent chilli pepper indigenous to Assam with tremendous potential for pharmaceutical applications. An attempt was made to standardize an efficient micropropagation protocol using silver nitrate (AgNO3 ) and glutamine as the key components alongside other plant hormones. In vitro propagation was carried out with seed and shoot tip explants. MS medium supplemented with different combinations of plant growth hormones (BAP, GA3 , NAA, and IBA), fortified with AgNO3 and glutamine was used for regeneration of Lota Bhot. Addition of AgNO3 and glutamine into the culture medium greatly improved proliferation in selected In vitro plantlets. A maximum number of multiple shots was recorded in plantlets derived from shoot tips. The process of flower induction and fruit formation In vitro were found to be more productive when treated with AgNO3 along with GA3 . IBA generated better results in the process of root initiation than NAA. The response of shoot tip explants towards In vitro processes was better than seed explants. Sterile soil enriched with vermicompost upgraded the chances of acclimatization of In vitro raised healthy plantlets.

Evaluation of the micropropagation potential of curauá pineapple hybrids for fiber production / Avaliação do potencial de micropropagação de híbridos do abacaxi curauá para a produção de fibras

Plant fiber is a renewable and biodegradable material that can be used effectively to reinforce various composites. Pineapple hybrids selected for their fiber quality are in the phase of agronomic validation in Brazil by the Embrapa Cassava and Fruits research unit. The selection of a hybrid for large-scale fiber production depends on obtaining a large number of seedlings. This study evaluated the morphogenetic response and propagation potential of eight hybrids of Ananas comosus var. erectifolius, for the purpose of producing high-quality seedlings on a large scale. Stem and crown buds were reduced and placed in MS nutritive medium supplemented with BAP at 0.5 mg L-1, NAA at 0.01 mg L-1 and Phytagel® at 2.5 g L-1. After 45 days, the number of oxidized, contaminated and surviving buds was determined. Swollen buds and plantlets were transferred to a multiplication medium containing MS sucrose, salts and vitamins. The propagation potential was evaluated based on the geometric growth rate among sub-cultures. The FIB-NEG hybrid presented the best results for the establishment phase (40.28%). The best propagative potential was obtained from crown buds with the highest values for FIB-EST (3.93), FIB-MIN (3.91) and FIB-BOY (3.91) hybrids.

A fibra vegetal é uma fonte renovável, biodegradável e de excelente desempenho como reforço em compósitos variados. Híbridos selecionados pela qualidade de suas fibras estão em fase de validação agronômica na Embrapa Mandioca e Fruticultura e sua adoção para produção de fibra em larga escala depende de um elevado número de mudas. Este trabalho teve como objetivo avaliar a resposta morfogenética e o potencial propagativo de oito híbridos de Ananas comosus var. erectifolius, com a finalidade de produzir mudas de qualidade em larga escala. Gemas do caule e coroa foram reduzidas, introduzidas em meio nutritivo MS suplementado com BAP a 0,5 mg L-1, ANA a 0,01 mg L-1 e Phytagel® a 2,5 g L-1. Aos 45 dias foram avaliados o número de gemas oxidadas, contaminadas e sobreviventes. Gemas intumescidas e plantas formadas foram transferidas para o meio de multiplicação contendo sacarose, sais e vitaminas MS. Avaliou-se o potencial propagativo a partir de uma taxa de crescimento geométrico entre subcultivos. O híbrido FIB-NEG (40.28%) apresentou os melhores resultados em porcentagem para a fase de estabelecimento. O melhor potencial propagativo foi obtido a partir de gemas de coroa, com os valores mais elevados registrados para os híbridos FIB-EST (3.93), FIB-MIN (3.91) e FIB-BOY (3.91).

Estrategias para disminuir la necrosis apical en la multiplicación y el enraizamiento in vitro del Pistacho / Strategies to reduce the apical necrosis in vitro multiplication and rooting of Pistachio

El pistacho (Pistacia sp.) es uno de los frutales menos explotados, entre las principales causas está el elevado costo del material vegetal por las dificultades de propagación de la especie. La propagación in vitro ofrece un gran potencial para la industria de esta especie por la multiplicación a gran escala de clones seleccionados. El objetivo del presente trabajo fue controlar la necrosis apical de los brotes en la propagación in vitro del pistacho. A partir de brotes jóvenes de plantas de esta especie mantenidas en casas de cultivo, se procedió a su desinfección con hipoclorito de sodio al 1% durante diferentes tiempos. Las yemas apicales y axilares se establecieron en el medio de cultivo Murashige & Skoog (1962) modificado y enriquecido con 1 mg/L de Metatopolina. Se evaluaron diferentes tipos de frascos, número y tipo de explantes y formulaciones de medios de cultivo durante dos subcultivos de multiplicación y en el enraizamiento. El frasco de cristal de 200 ml de capacidad, la línea prolifera y el medio de cultivo DKW lograron los valores más bajo de necrosis apical. En la fase de multiplicación se presentaron valores bajo en todos los tratamientos ensayados sin diferencia estadísticas entre ellos en comparación con el enraizamiento donde la necrosis apical alcanzó valores superiores. El medio DKW y las líneas prolíferas lograron los menores valores. El medio de cultivo MS modificado, favoreció el número de segmentos enraizados y el DKW (Driver & Kuniyuki, 1984) el número de segmentos que brotaron, presentando este último menor incidencia de necrosis apical.

The pistachio (Pistacia sp.) is one of the least exploited fruit among the main causes is the high cost of plant material by the difficulties of propagation of the species. The propagation in vitro offers great potential for the industry of this species by multiplication scale of selected clones. The aim of this study was to control the apical necrosis of outbreaks in the in vitro propagation Pistachio. From young shoots of plants of this species kept in growing houses, proceeded to disinfection with sodium hypochlorite 1% for different times. The apical and axillary buds were established in the culture medium Murashige and Skoog (1962) modified and supplemented with 1 mg/L Metatopolina. Different types of bottles, number and type of explants and culture media formulations for two subcultures multiplication and rooting were evaluated. Glass flask of 200 ml capacity, and line proliferates DKW medium achieved the lowest values apical necrosis. In the multiplication phase values were low in all treatments tested no statistical difference between them compared to rooting where apical necrosis reached higher values. The average DKW and prolific lines obtained lower values. MS medium modified crop, favored the number of segments rooted and DKW (Driver and Kuniyuki, 1984) the number of segments that sprouted, the latter having lower incidence of apical necrosis.

The effect of spectral light quality on in vitro culture of sugarcane / Efeito da qualidade espectral da luz no cultivo in vitro de cana-de-açúcar

The effects of different light quality treatments on in vitro growth and multiplication of sugarcane (RB867515) were investigated. The plantlets were cultivated on MS medium containing 1.3 µM 6-benzylaminopurine (BAP), and exposed to five light treatments: three combinations of blue/red LED (70:30, 50:50, 30:70), white-LED and fluorescent lamps, during 24 days. Among the LED light treatments, blue/red combination in 50:50 proportions proved to have the best results for stem length, fresh mass, leaf number and shoot multiplication. Higher content of photosynthetic pigments was also obtained with LEDs. Results suggested that the light quality emitted by LEDs was suitable for plant growth and development and it may be used as alternative and economic light source for micropropagation of sugarcane variety under analysis.

Foram investigados os efeitos de diferentes tratamentos de qualidade da luz sobre o crescimento e multiplicação in vitro de cana-de-açúcar (RB867515). As plantas foram cultivadas em meio MS contendo 1,3 µ M de 6-benzilaminopurina (BAP) e expostas a cinco tratamentos de luz: três combinações de LED azul/vermelho (70:30, 50:50, 30:70), LED branco e lâmpadas fluorescentes, durante 24 dias. Entre os tratamentos de luz de LED, a combinação azul/vermelho na proporção 50:50 apresentou os melhores resultados para altura, massa fresca, número de folhas e multiplicação de brotos. Maior teor de pigmentos fotossintéticos também foi obtido com a utilização de LEDs. Os resultados sugerem que a qualidade da luz emitida pelos LEDs foi adequada para o crescimento e desenvolvimento das plantas e pode ser utilizada como fonte de luz alternativa e econômica para a micropropagação da variedade de cana-de-açúcar estudada.

In vitro development of Encyclea cordigera in different concentrations of honey / Desenvolvimento in vitro de Encyclea cordigera em diferentes concentrações de mel

ABSTRACT: The objective of this paper was to evaluate the effects of different honey concentrations in culture media, in comparison to sucrose medium, for the in vitro development of the epiphytic Encyclea cordigera orchid, in order to improve the process of propagation of the species. The in vitro germination was prepared on a reduced Murashige & Skoog (MS) medium. After 90 days, the seedlings were divided into different treatments, where they remained for another 90 days. Six treatments were set up (30g L-1 of sucrose; 15, 30, 45, and 60g L-1 of honey; and absence of any carbohydrates) in a completely randomized design. Plants were removed from the vials 270 days after the start of the experiment, and the number of roots, length of the largest leaf, length of the longest root, number of leaves, and fresh and dry masses were evaluated. Data concerning the number of leaves and roots were (x+1)1/2 transformed and subjected to an analysis of variance (ANOVA); the means were compared by a Tukey's test set at 5% probability. Medium containing 60g L-1 of honey proved to be superior to the sucrose medium traditionally used, favoring the in vitro growth and development of Encyclea cordigera. This medium can therefore be recommended for the propagation of this species, which is usually cultivated as an ornamental plant.

RESUMO: Este trabalho foi realizado com objetivo de avaliar a influência da concentração de mel no meio de cultura, comparando-o com a sacarose, para o desenvolvimento in vitro da orquídea epífita Encyclea cordigera, a fim de aperfeiçoar o processo de propagação dessa espécie. A semeadura in vitro procedeu-se em meio de cultura Murashige & Skoog (MS) reduzido e, após 90 dias, as plântulas foram distribuídas entre os tratamentos, em que permaneceram por mais 90 dias. Seis tratamentos (30g L-1 de sacarose, 15, 30, 45 e 60g L-1 de mel e ausência de carboidrato) foram utilizados, em delineamento inteiramente casualizado. Após 270 dias do início do experimento, as plantas foram retiradas dos frascos, sendo avaliado o número de raízes, comprimento da maior folha, comprimento da maior raiz, número de folhas, massa fresca e massa seca. Os dados de número de folhas e de raízes foram transformados (x+1)1/2 e submetidos à análise de variância (ANAVA); as médias foram comparadas pelo teste de Tukey a 5% de probabilidade. O meio de cultura suplementado com 60g L-1 de mel favoreceu o crescimento e desenvolvimento in vitro de Encyclea cordigera, sendo superior ao uso de sacarose, que é tradicionalmente usado, podendo ser recomendado para propagação dessa orquídea, que apresenta potencial ornamental.

EFFECT OF GENOTYPE ON THE in vitro REGENERATION OF Stevia rebaudiana VIA SOMATIC EMBRYOGENESIS / Efecto del genotipo sobre la regeneración in vitro de Stevia rebaudiana a través de embriogénesis somática

Stevia rebaudiana (Asteraceae) is a plant of economic importance because of its medicinal properties and the presence of sweetener compounds on its leaves. These compounds can be a substitute for sucrose in a wide variety of products used by persons with diabetes and obesity problems. To standardize an efficient and effective propagation method for the different Stevia genotypes grown in Colombia, this study evaluated the effect of different combinations of the plant growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 6-(gamma, gamma-dimethylallylamino) purine (2iP) and Zeatin on the induction and development of somatic embryos. Adenine and coconut water were also evaluated as supplements in the basal culture medium Murashige and Skoog Basal Salt Mixture (MS) with glutamine. The combination of 2,4-D (18.09 µM) and 2iP (7.38 µM) produced the highest number of somatic embryos per explant, which had well-defined characteristics. The genotype showed a significant effect on the embryogenic response. In the "SRQ-93" genotype, the formation and development of somatic embryos was achieved, whereas the genotypes "Bertoni" and "Morita II" only yielded embryogenic and non-embryogenic calli, respectively. The conversion to seedlings was achieved on the regeneration medium containing gibberellic acid (GA3) (0.29 µM) and activated charcoal.

Stevia rebaudiana (Asteraceae), es una planta de gran importancia económica debido a sus propiedades medicinales y a la presencia de compuestos endulzantes en sus hojas, los cuales pueden sustituir la sacarosa en gran variedad de productos utilizados por personas con problemas de diabetes y obesidad. Con el propósito de estandarizar un método de propagación eficiente y efectivo para diferentes genotipos de Stevia cultivados en Colombia, en la presente investigación se evaluó el efecto sobre la inducción y desarrollo de embriones somáticos de diferentes combinaciones de los reguladores de crecimiento vegetal 2,4-D, IAA, IBA, 2iP y Zeatina, además de los suplementos adenina y agua de coco en el medio de cultivo basal Murashige y Skoog (1962), adicionado con glutamina. Con la combinación 2,4-D (18.09 µM) y 2iP (7.38 µM) se obtuvo el mayor número embriones somáticos por explante con características bien definidas. El genotipo tuvo un efecto significativo en la repuesta embriogénica, en el genotipo "SRQ-93" se logró la formación y el desarrollo de embriones somáticos, mientras que en los genotipos "Bertoni" y "Morita II", solo se obtuvo callo embriogénico y no embriogénico respectivamente. La conversión a plántulas se alcanzó en el medio de regeneración conteniendo GA3 (0.29 µM) y carbón activado.

Assessment of mint (Mentha spp) species for large-scale production of plantlets by micropropagation / Avaliação de espécies de menta (Mentha spp) para a produção de mudas em larga escala por micropropagação

Species of the genus Mentha produce essential oils which are widely used in pharmaceutical and cosmetic industries. Current study evaluates the potential for in vitro propagation and estimates mass production of plantlets of Mentha species. Nine species (M. piperita, M. suaveolens, M. canadensis, M. longiflora, M. aquatica, M. arvensis, Mentha x gracilis, M. gracilis and M. spicata) were propagated with five successive 30-day subcultures in MS medium supplemented with NAA (0.05 µM) and BAP (4.4 µM). Shoots were rooted in MS with IBA, IAA or NAA (0.0; 0.25; 0.5; 2.5 or 5.0 µM). The rooted plantlets were finally acclimatized in a greenhouse. Studied species increased in multiplication rates between 4.2 and 9.0-fold per month. M. piperita, M. longiflora, M. arvensis, M. x gracilis and M. gracilis showed the greatest potential for plantlet production since the estimated production varied between 6,000 and 27,000 plantlets after five 30-days subcultures. The addition of auxin to the medium did not influence root induction. However, IAA at a concentration of 5 µM provided the best results for root length and fresh weight, with averages 11.1 cm and 0.16 g, respectively. Survival of plantlets reached 100% during the greenhouse acclimatization process.

Espécies do gênero Mentha produzem óleos essenciais largamente usados na indústria farmacêutica e de cosméticos. O estudo avaliou o potencial de propagação in vitro e estimou a produção de mudas de espécies de menta. Nove espécies (M. piperita, M. suaveolens, M. canadensis, M. longiflora, M. aquatica, M. arvensis, Mentha x gracilis, M. gracilis e M. spicata) foram propagadas por até cinco sucessivos subcultivos de 30 dias em meio de MS adicionado de ANA (0,05 µM) e BAP (4,4 µM). Os brotos foram enraizados em meio de MS com AIB, AIA ou ANA (0,0; 0,25; 0,5; 2,5 ou 5,0 µM). Finalmente, as mudas foram aclimatizadas em casa de vegetação. As espécies estudadas apresentaram aumentos nas taxas de multiplicação, variando entre 4,2 e 9,0 vezes por mês. M. piperita, M. longiflora, M. arvensis, M. x gracilis e M. gracilis mostraram os melhores potenciais para propagação, uma vez que a produção variou entre 6.000 e 27.000 mudas após cinco subcultivos de 30 dias. A adição de auxina no meio não influenciou a indução de raízes. Entretanto, o AIA na concentração de 5 µM promoveu os melhores resultados quanto ao comprimento e massa fresca das raízes, com médias de 11,1 cm e 0,16 g, respectivamente. No processo de aclimatização houve 100% de sobrevivência das mudas.

Micropropagation of Pluchea sagittalis (Lam. ) Cabrera / Micropropagação do quitoco (Pluchea sagittalis (Lam. ) Cabrera)

ABSTRACT: The objective of this study was to develop an in vitro protocol for the micropropagation of Pluchea sagittalis (Lam.) Cabrera. Plants were regenerated in vitro from stem segments. The procedure employed includes: 1) surface sterilization of shoots by immersion in 70% ethanol for 10 s followed by 1.0% NaOCl for 10 min, and subsequent immersion in 0.05% HgCl2 for 3 min and two washes with sterile distilled water; 2) induction of root and shoot by culture on hormone-free Murashige and Skoog medium (MS); 3) acclimatization of 60 day-old-plantlets in soil under ex vitro conditions. Minimum contamination was observed for apical shoot explants (10%). However, independently of the explant position in the stem, all explants regenerated new shoots. Various successive cultivations from stem explants every 60 days during more than 1 year have been shown to be a suitable method to propagate P. sagittalis in vitro. Low salt concentration (25% of the normal concentration) in the medium promoted greater growth of plantlets because the plants had a higher number of roots and longer roots in such an environment. Our protocol for the micropropagation of P. sagittalis can be accomplished as a two-step procedure within a short period of time (two months) before transplanting.

RESUMO: O Objetivo deste estudo foi desenvolver um protocolo para a micropropagação in vitro da Pluchea sagittalis (Lam.) Cabrera. Plantas foram regeneradas in vitro a partir de segmentos de ramo. O procedimento empregado incluiu: 1) esterilização da superfície de ramos pela imersão em etanol 70% por 10 s seguida pela de NaOCl 1.0% por 10 min e, subsequentemente, em HgCl2 0.05% por 3 min e duas lavagens em água destilada e esterilizada; 2) indução de raízes e parte aérea pelo cultivo em meio Murashige & Skoog (MS) isento de hormônio; 3) aclimatização de plantas com 60 dias de idade em solo sob condições ex vitro. Contaminação mínima foi observada em explantes caulinares do ápice (10%). Entretanto, independentemente da posição do segmento no caule, todos explantes regeneraram novos ramos. Vários cultivos sucessivos a cada 60 dias durante mais de um ano tem mostrado ser um método adequado para a propagação in vitro de P. sagittalis. A baixa concentração de sais no meio (25% da concentração normal) promoveu maior crescimento das plântulas devido às mesmas apresentarem maior número e comprimento de raízes. O protocolo para a micropropagação da P. sagittalis pode ser executado em procedimento de duas etapas dentro de um período de tempo curto (dois meses) antes do transplantio.

Micropropagation of Brasilidium forbesii (Orchidaceae) through transverse and longitudinal thin cell layer culture / Micropropagação de Brasilidium forbesii (Orchidaceae) pela técnica 'thin cell layer' longitudinal e transversal

An efficient in vitro propagation method was established for Brasilidium forbesii (Hook.) Campacci using transverse and longitudinal thin cell layer (tTCL and lTCL, respectively) culture systems. Six-month-old protocorms from in vitro germinated seeds were used for this study. TCLs (1.0-mm thick) from protocorms were grown on Woody Plant Medium (WPM) supplemented with benzyladenine (BA) (0.5­4.0 µM).The lTCL technique was more efficient for inducing protocorm-like bodies (PLBs) and regenerating shoots than the tTCL technique. The frequency of PLB formation was influenced by BA concentration, and the lTCL explant grown on a medium containing 2.0 µM BA produced the highest percentage of new protocorms (77%) with a total of 22.7 PLBs per explant, after the first subculture on the same medium. Plantlet development was optimal on WPM medium containing 3.0 g L -1 activated charcoal, and indole-3 -butyric acid was not necessary for rooting. Regenerated plants were successfully acclimatized in a greenhouse after 16 weeks using vermiculite as the substrate (100% survival).

Foi estabelecido um método eficiente de propagação in vitro para Brasilidium forbesii (Hook.) Campacci utilizando a técnica 'thin cell layer' transversal (TCLt) e longitudinal (TCLl). Foram utilizados protocormos de seis meses obtidos da germinação in vitro. TCLs (1,0 mm de espessura) dos protocormos foram cultivados no meio 'Woody Plant Medium' (WPM), acrescido com benziladenina (BA) (0,5 a 4,0 µM). A técnica TCLl foi mais eficiente para indução de estruturas semelhantes a protocormos (ESPs) e regeneração de brotações do que a técnica TCLt. A frequência de formação de ESP foi influenciada pela concentração de BA e o explante TCLl, cultivado em um meio contendo 2,0 µM BA, produziu a mais alta percentagem de novos protocormos (77%), com um total de 22,7 PLBs por explante, após o primeiro subcultivo para o mesmo meio. O desenvolvimento das plântulas foi eficiente no meio WPM contendo 3,0 g L -1 de carvão ativado e o ácido indol-3- butírico (AIB) não foi necessário para o enraizamento. Plantas regeneradas foram estabelecidas com sucesso em casa de vegetação, utilizando vermiculita como substrato (100% de sobrevivência), após 16 semanas.

Enhanced in vitro Seedling Recovery in Nothapodytes nimmoniana (J. Graham) Mabberly.

Nothapodytes nimmoniana is an endangered medicinal tree and regarded most convenient natural source for large scale isolation of anticancer alkaloid camptothecin. In order to meet pharmaceutical market demand for camptothecin and conserve natural population of the species, in vitro culture techniques can be employed for mass propagation and production of the alkaloid. Seed germination is one of the difficult challenges in the establishment of the aseptic in vitro cultures. Pre-germination treatments of soaking in water, GA3 solution and aseptic excision of the embryo axis were employed to enhance seed germination and seedling recovery respectively for further in vitro studies. Seed soaking in water or GA3 solution for 24 hours enhanced the germination capacity with better response obtained when seeds were soaked in GA3 (2.6 μM) solution and germinated on half strength MS medium supplemented with the GA3. Removal of the outer, inner seed coat and inoculation of the embryo axis on medium amended with or without GA3 reduced seedling recovery time to 3-4 weeks. Seedlings obtained on medium fortified with GA3 showed vigorous growth with complete plantlet development than on PGR-free medium. The strategy can be employed to reduce seedling recovery time to within 3-4 weeks compared to 7-10 weeks required for germinating the seeds in vitro.

A simple technique for tracking individual spore and gametophyte development in Adiantum lunulatum Burm. f. using modified extra thin alginate film technique.

A new technique was developed for accurate calculation of percent germination and tracking of individual spores from germination to gametophyte development in Adiantum lunulatum. High percentage of ETAF immobilized spore germination (72.4%) was followed by development of gametophytic clumps. The ETAF immobilized clumps were cut into pieces and multiplied en masse. Apomictic sporophytes developed from the gametophytes. This indicated the potential of ETAF for mass propagation of A. lunulatum without the need to start from spores. Since individual spores can be tracked from germination to gametophyte development, the ETAF technique has the potential to be used for (i) harvesting uniformly developed plants of similar age for extensive experimentations and commercial utilization and (ii) detailed study on developmental and reproductive biology of different ferns and fern allies.

In vitro germination and acclimatization of cambui tree type seedlings / Germinação in vitro e aclimatização de tipos de cambuizeiro

There are few reports in literature on the in vitro behavior of cambui tree (Myrciaria tenella O. Berg) and acclimatization conditions. The aim of this study was to evaluate the effect of culture media on in vitro germination and the effect of different substrates on the acclimatization of two Myrciaria tenella types. The study was carried out at the Embrapa Tabuleiros Costeiros Laboratory of Plant Tissue Culture, Aracaju, SE. Seeds were extracted from fruits of two Myrciaria tenella types: Orange and Purple Types. The seeds were inoculated in the following culture media: T1 - MS medium + 30g L -1 sucrose, T2 - 1/2 MS medium + 15g L -1 sucrose and T3 - control without MS salts. To study the effect of substrates on acclimatization, seedlings were transferred to plastic containers with capacity of 300cm 3 containing the following sterilized substrates: S1 - soil and powdered coconut husk - SPC (1:1 by volume); S2 - soil, washed sand and powdered coconut husk - SAPC (1:1:1 by volume) and S3 - Biomix (r) commercial substrate - SC. The medium without MS salts promoted 100% in vitro germination and 1/2 MS medium greater development of seedlings. All substrates studied are suitable for acclimatization of seedlings germinated in vitro. Myrciaria tenella of yellow type showed greater vigor during acclimatization. .

São poucos os relatos na literatura sobre estudos do comportamento do cambuizeiro (Myrciaria tenella O. Berg) in vitro e condições de aclimatação. O objetivo do trabalho foi de avaliar o efeito de meios de cultura na germinação in vitro e o efeito de diferentes substratos na aclimatação de tipos de cambuizeiros. O estudo foi realizado no Laboratório de Cultura de Tecidos de Plantas da Embrapa Tabuleiros Costeiros, Aracaju, SE. Foram utilizadas sementes extraídas de frutos de dois tipos de cambuizeiros: Tipo Laranja e Tipo Roxo. Para o estudo de meios para a germinação, as sementes foram inoculadas nos seguintes meios de cultura: T1 -meio de MS + 30g L -1 de sacarose, T2 - meio 1/2 MS + 15g L -1 de sacarose e T3-testemunha sem sais MS. Para o estudo do efeito de substratos na aclimatação, as plântulas foram transferidas para recipientes plásticos com capacidade de 300cm 3 , contendo os seguintes substratos esterilizados: S1 - solo e pó de casca de coco seco - SPC (1:1, em volume); S2 -solo, areia lavada e pó de casca de coco seco - SAPC (1:1:1, em volume); e S3-substrato comercial Biomix (r) - SC. O meio sem sais MS proporciona 100% porcentagem de germinação in vitro e o meio 1/2 MS maior desenvolvimento em altura de plântulas. Todos os substratos estudados são adequados para aclimatação de plântulas germinadas in vitro. O cambuizeiro do tipo Amarelo apresenta maior vigor na aclimatação. .

Large scale propagation of an exotic edible bamboo, Phyllostachys pubescens Mazel ex H. De Lehale (Moso Bamboo) using seeds.

For ex vitro propagation, seeds of P.pubescens were treated with different concentrations of gibberellic acid (GA3) and germination of seeds was tested both in plastic pots as well as by direct sowing in the nursery beds. Maximum seed germination was achieved when treated with 200 mgL–1 (w/v) GA3. For in vitro propagation, an exposure of nodal explants from in vitro raised seedlings to 0.2 mgL–1 1–phenyl–3–(1,2,3–thiadiazol–5–yl) urea and 1 mgL–1 kinetin supplemented medium for 30 days and thereafter to hormone free Murashige and Skoog basal medium resulted in axillary shoot proliferation. For rooting, in vitro raised shoots were exposed to MS medium containing 2 mgL–1 indole-3-butyric acid for 15 days and then shifted to hormone free medium. On an average, 2.8 shoots were obtained in 75% of the cultures within 4 weeks. Such in vitro raised plants were successfully hardened and shifted to field conditions.

Pigment production and growth of Alternanthera plants cultured in vitro in the presence of tyrosine

The aim of the present study was to investigate the influence of tyrosine on the in vitro growth and the production of the betacyanin pigment in Alternanthera philoxeroides and A. tenella. Nodal segments were inoculated in MS medium containing different concentrations of tyrosine (0, 25, 50 and 75 μM), and the number of sprouts and buds, height, root length, fresh matter of shoots and roots and betacyanin content were evaluated. In A. philoxeroides , the highest production of betacyanin (51.30 mg 100 g-1 FM) was in the stems with the addition of approximately 45 μM tyrosine, while the increase in the leaves was proportional to the tyrosine concentration, and the best average was obtained with a tyrosine concentration of 75 μM (15.32 mg 100 g-1 FM). Higher tyrosine concentrations were deleterious to the growth of A. tenella plants, and a concentration of 75 μM was considered toxic. However, a tyrosine concentration of 50 μM benefitted betacyanin production, which reached 36.5 mg 100 g-1 FM in the plant shoots. These results showed the positive effect of tyrosine on the production of betacyanin in both species; however, application at high concentrations hampered the growth of Alternanthera plants.

In vitro Propagation and GC-MS Studies of Ocimum basilicum Linn. var. pilosum (Willd.) Benth.

An in vitro propagation method is outlined for Ocimum basilicum Linn. var. pilosum (Willd.) Benth., a wild aromatic plant belongs to Lamiaceae family. Shoot buds were used as source of explants on MS media supplemented with different concentrations of growth regulators for callus growth, induction of multiple shoots and roots respectively. MS media with 1.5 mg/L of kinetin and 0.5 mg/L of NAA showed 95.5% shooting, maximum number of shoots (7.33) and relatively better shoot lengths (4.15 cm). Excised shoots were carefully transferred to halfstrength MS medium supplemented with 1.0 mg/L indole-3-butyric acid (IBA) for root induction and it yields 86.6% rooting. Whereas, average root length and number of roots observed were 1.73 cm and 3.31 respectively per explants. Rooted plantlets were hardened and successfully established in natural soil, where they grew and matured normally. GC-MS studies in methanolic leaf extract of naturally grown Ocimum species yielded 15 compounds. Two compounds viz. cis-9-Hexadecenal (35.06%) and n-Hexa decanoic acid (21.6%) accounted for the major share (56.66%). On the other hand, 2-hydroxy-6-methylbenzaldehyde (10.99%) as well as 4H-1-Benzopyran-4-one and 5-Hydroxy-6, 7-Dimethoxy-2-(4- Methoxyphenyl) (7.75%) represented only 18.74%. Establishment of reliable in vitro propagation protocol, with phytochemical profile of hitherto unreported Ocimum species further widens the scope to evaluate its therapeutic properties.

Progress in micropropagation of Passiflora spp. to produce medicinal plants: a mini-review

Micropropagation of Passiflora species and its hybrids may play an important role in the production of healthy and disease-free plants which can be a source of medicinal herbal products, nutritional fruits and ornamental flowers. The rapid multiplication of elite plants to obtain pharmacognostic material, containing valuable flavonoid C-glycosides, is possible by usingcontrolled in vitro conditions, constituents of the medium and the interactions of plant growth regulators (1-naphtaleneacetic acid, benzyladenine, gibberellin GA3,kinetin, indole-3-acetyl-L-aspartic acid, indole-3-butyric acid, thidiazuron) and influencing various chemical additives (silver nitrate, coconut water, activated charcoal). Investigations of specific requirements during stages of micropropagation, such as the establishment of primary cultures (including type of explants, age of donor plant), shoot multiplication (by direct and indirect organogenesis and embryogenesis), rooting and acclimatization of regenerated plants are summarized in this review. The following species were recently studied for micropropagation: P. alata, P. caerulea, P. cincinnata, P. edulis, P. foetida, P. setacea, P. suberosa. It seems that for awide range of applications of in vitro clones of Passiflora, interdisciplinary studies including genetic and phytochemical aspects are needed.